Whole-genome synthesis and characterization of viable S13-like bacteriophages.
Whole-genome synthesis and characterization of viable S13-like bacteriophages.
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活性 S13 样噬菌体的全基因组合成和表征
DOI：
10.1371/journal.pone.0041124
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Cai Z
中科院分区：
文献类型：
--
作者：
Liu Y;Han Y;Huang W;Duan Y;Mou L;Jiang Z;Fa P;Xie J;Diao R;Chen Y;Ye Y;Yang R;Chen J;Sun X;Li Z;Tang A;Gui Y;Cai Z
背景高通量DNA测序和从头合成技术取得了前所未有的进步，使我们能够在没有天然模板的情况下创造出活的有机体。方法/主要发现野生型S13噬菌体基因组序列从GenBank下载。将两个同义突变引入wt-S13基因组，产生m1-S13基因组。通过对wt-S13基因组衣壳蛋白编码区的两个非同义突变进行改造，获得了另一个突变体m2-S13基因组。用噬菌体G4的F衣壳蛋白ORF取代噬菌体S13的F衣壳蛋白开放阅读框(ORF)，构建了嵌合的噬菌体基因组。所有四个噬菌体的整个基因组都是由一系列化学合成的短重叠寡核苷酸组装而成的。线性合成的基因组被环化并电穿孔到S13噬菌体的标准实验室宿主E.ColiC中。所有四个噬菌体都被回收，并显示出斑块。测序结果表明，这些合成的基因组是准确的。合成的噬菌体能够裂解其细菌宿主，并能耐受一般环境条件。在含CaCl2的LB培养基中，变异菌株间的表型差异不明显，但S13/G4嵌合体对缺钙更为敏感，在无钙条件下具有较低的吸附速率。结论/意义噬菌体S13及其变异体可以通过化学方法合成。噬菌体G4的主要衣壳基因在噬菌体S13的生命周期中起作用。这些结果支持一种进化假说，该假说认为微病毒科的历史中应该包括一个涉及差异很大的祖先谱系的F基因的同源重组事件。
Background Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template. Methodology/Principal Findings The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl2, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition. Conclusions/Significance The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.
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影响因子：
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作者：
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通讯作者：
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